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Title

Author

Defense Date

2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Microbiology & Immunology

First Advisor

Dr. Janina P. Lewis

Abstract

Manganese and iron homeostasis play an important role in oxidative stress protection in a variety of organisms. However, the transport and role of these metals in the periodontal pathogen Porphyromonas gingivalis were not well understood. Analysis of the genome of P. gingivalis W83 revealed the presence of two genes encoding homologs of ferrous iron transport protein, FeoB1 and FeoB2. The goal of this study was to determine the role of these two putative transporters in metal transport, their contributions to resistance to oxygen radicals and intracellular survival as well as the regulation and genetic organization of these two loci. Isogenic mutant strains deficient in FeoB1 and FeoB2, respectively, were generated and used in this study. The transport ability for manganese and iron was assessed and compared in feoB1, feoB2 mutant and wild type strains using 55Fe2+ and 54Mn2+. We demonstrated that feoB2 encodes a major manganese transporter, while FeoB1 functions as a major ferrous iron transporter. The roles of P. gingivalis FeoB1 and FeoB2 in oxidative stress defense and intracellular survival in host cells were determined using an oxidative stress survival assay and an in vitro infection assay, respectively. The feoB2 mutant exhibited reduced survival after exposure to H2O2 and to atmospheric oxygen and inside the host cells compared to the wild-type strain and its revertant, while the feoB1 mutant survived as well as the wild type strain under oxidative stress and possessed better capability to adhere to and survive in the host cells. Our results demonstrate that FeoB2 is required for protection of the bacterium from oxidative stress and for intracellular survival of P. gingivalis in host cells. However, FeoB1 is dispensable for both processes. Quantitative RT-PCR analysis revealed that expression of feoB2 in P. gingivalis is induced by oxidative stress. However, expression of feoB1 increased 2-fold upon exposure to lower growth temperature. Both observed inductions were specific and not detected under other stress conditions. We have also showed in this study that feoB2 is the second gene transcribed in an operon that is composed of a total of five genes and feoB1 is only co-transcribed with one downstream gene encoding a hypothetical protein. Notably, we also identified tandem repeats with potential to form stable stem-loop RNA secondary structure within the feoB2 and feoB1 transcripts.To our knowledge, this study has demonstrated the first connection among metal homeostasis, oxidative stress resistance and response to host cells in the periodontal pathogen, P. gingivalis.